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PreprintCited by 83Advanced Proteomics Techniques and ApplicationsMass Spectrometry Techniques and ApplicationsMetabolomics and Mass Spectrometry Studies

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Fast and Deep Phosphoproteome Analysis with the Orbitrap Astral Mass Spectrometer

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Lancaster, Noah M., Sinitcyn, Pavel, Forny, Patrick, Peters-Clarke, Trenton M., Fecher, Caroline, Smith, Andrew J., Shishkova, Evgenia, Arrey, Tabiwang N., Pashkova, Anna, Robinson, Margaret Lea, Arp, Nicholas, Fan, Jing, Hansen, Juli, Galmozzi, Andrea, Serrano, Lia R., Rojas, Julie, Gasch, Audrey P, Westphall, Michael S., Stewart, Hamish, Hock, Christian, Damoc, Eugen, Pagliarini, David J., Zabrouskov, Vlad, Coon, Joshua J.·2023·Nature Communications
DOI 10.1101/2023.11.21.568149PMC PMC11327265

Preprint — published as 10.1038/s41467-024-51274-0

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Abstract

Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. The technology was benchmarked to other state-of-the-art MS platforms using both synthetic peptide standards and with EGF-stimulated HeLa cells. We applied this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detected 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence, structural, and kinase specificity context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of novel phosphorylation events relevant to mitochondrial and brain biology.

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Metadata

Authors
Lancaster, N. M., Sinitcyn, P., Forny, P., Peters-Clarke, T. M., Fecher, C., Smith, A. J., Shishkova, E., Arrey, T. N., Pashkova, A., Robinson, M. L., Arp, N., Fan, J., Hansen, J., Galmozzi, A., Serrano, L. R., Rojas, J., Gasch, A. P., Westphall, M. S., Stewart, H., Hock, C., Damoc, E., Pagliarini, D. J., Zabrouskov, V., & Coon, J. J.
Affiliations
  • University of Wisconsin–Madison (US)
  • Morgridge Institute for Research (US)
  • Washington University in St. Louis (US)
  • Thermo Fisher Scientific (Germany) (DE)
  • Thermo Fisher Scientific (United States) (US)
Corresponding author
Coon, Joshua J.
ORCID
Venue
Nature Communications
Publisher
openRxiv
Published
2023-11-21
Volume
15
Issue
1
Pages
7016-7016
ISSN
2041-1723 (ISSN-L)
DOI
10.1101/2023.11.21.568149
PMC
PMC11327265
OpenAlex
W4401615298
Cited by
83
References
1
Citations by year
2026: 34 · 2025: 41 · 2024: 8
FWCI
10.35
Type
journal article
Language
en
Open access
Licence
cc-by-nc-nd
Locations
  • Nature Communications (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by-nc-nd)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion, cc-by-nc-nd)
  • PubMed Central (submittedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • National Science Foundation (R35GM118110, DE-SC0018409, 2010789)
  • U.S. Department of Energy (DESC0018409)
  • U.S. Department of Health and Human Services (R35GM118110, P41GM108538)
  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (211038)
  • National Institutes of Health
  • ACS Division of Analytical Chemistry
  • Great Lakes Bioenergy Research Center (DE-SC0018409)
  • National Human Genome Research Institute (T32HG002760)
  • National Institute of General Medical Sciences (T32GM135066, R35GM118110, P41GM108538, R35GM150899, R35GM147014)
  • National Institute of Diabetes and Digestive and Kidney Diseases (R01DK098672)
Topics
  • Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
  • Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
Keywords
phosphoproteomics, protein phosphorylation, data-independent acquisition, phosphorylation site mapping, kinase specificity, mitochondrial phosphorylation

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